PROJECT SUMMARY/ABSTRACT Cerebral microhemorrhages (CMH) result from rupture of small intracerebral blood vessels and progressively impair neuronal function. The incidence of CMH dramatically increases with age and hypertension and is a major cause for age-related cognitive decline. Cognitive decline caused by CMH has severe impacts on quality-of-life, yet remains untreatable. CMHs occur due to increased vascular fragility but underlying mechanisms are unknown, and thus therapeutic interventions to mitigate CMHs are not available. Blood vessel integrity requires plasticity of vascu- lar smooth muscle cells (VSMCs), which exhibit an adaptive switch from a highly contractile to a protective anti- fragility phenotype in response to stress. Aging fundamentally alters VSMC phenotypic switching, suppressing the adoption of this protective VSMC phenotype. In contrast, insulin-like growth factor (IGF)-1 has vasoprotective ef- fects and promotes adoption of the protective anti-fragility phenotype, but circulating IGF-1 levels are dramatically decreased with age. Low IGF-1 levels increase the risk for cerebromicrovascular disease and promote the devel- opment of CMH in our rodent models, supporting a role for IGF-1 deficiency in age-related vascular fragility. Our hypothesis is that impaired VSMC phenotypic switching due to IGF-1 deficiency has a fundamental role in in- creased cerebrovascular fragility and development of CMH with age. Aim 1 will test the hypothesis that in- creased CMH in aging is due to vascular fragility arising from decreased IGF-1 signaling in VSMCs. Development of CMH, associated neurological/gait defects, and VSMC phenotype will be compared in mice with VSMC-specific disruption of IGF-1 signaling, mice with overall disruption of IGF-1 signaling, and aged mice coupled with IGF-1 supplementation/rescue. Aim 2 will use cultured aged, young, and IGF-1 receptor knockdown VSMCs to test the hypothesis that IGF-1 signaling is required for VSMC cellular adaptation to hemodynamic stress and will eval- uate novel mechanisms mediated by the transcription factors Tbx15/18 for this regulation. Aim 3 will test the hypothesis that VSMC phenotypic heterogeneity influences the development of CMH. VSMCs exist in a heter- ogeneous pool in which multiple phenotypes co-exist, determined based on growth factors including IGF-1 and other stimuli in the extracellular space, but the characteristics of this heterogeneity in cerebral arteries is un- known. VSMC lineage tracing genetic mouse models of aging and IGF-1 deficiency, coupled with single-cell RNA-sequencing will be used to characterize VSMC heterogeneity. Newly discovered pro- and anti-fragility VSMC phenotypic states will be correlated with location of CMH bleeds to test the hypothesis that CMHs occur primarily in regions where VSMCs show a maladaptive phenotype The scientifically and technically innovative studies proposed here will significantly enhance our understanding of the role of IGF-1 deficiency in the devel- opment of CMH and will provide critical insight into cellular mechanisms underlying it, both of which are critical for the development of effective therapies.